Impact of distinct FG nucleoporin repeats on Nup98 self-association.

Nucleoporins rich in phenylalanine/glycine (FG) residues form the permeability barrier within the nuclear pore complex and are implicated in several pathological cellular processes, including oncogenic fusion condensates. The self-association of FG-repeat proteins and interactions between FG-repeats play a critical role in these activities by forming hydrogel-like structures. Here we show that mutation of specific FG repeats of Nup98 can strongly decrease the protein's self-association capabilities. We further present a cryo-electron microscopy structure of a Nup98 peptide fibril with higher stability per residue compared with previous Nup98 fibril structures. The high-resolution structure reveals zipper-like hydrophobic patches which contain a GLFG motif and are less compatible for binding to nuclear transport receptors. The identified distinct molecular properties of different regions of the nucleoporin may contribute to spatial variations in the self-association of FG-repeats, potentially influencing transport processes through the nuclear pore.

Multimodal interactions drive chromatin phase separation and compaction.

Gene silencing is intimately connected to DNA condensation and the formation of transcriptionally inactive heterochromatin by Heterochromatin Protein 1α (HP1α). Because heterochromatin foci are dynamic and HP1α can promote liquid-liquid phase separation, HP1α-mediated phase separation has been proposed as a mechanism of chromatin compaction. The molecular basis of HP1α-driven phase separation and chromatin compaction and the associated regulation by trimethylation of lysine 9 in histone 3 (H3K9me3), which is the hallmark of constitutive heterochromatin, is however largely unknown. Using a combination of chromatin compaction and phase separation assays, site-directed mutagenesis, and NMR-based interaction analysis, we show that human HP1α can compact chromatin in the absence of liquid-liquid phase separation. We further demonstrate that H3K9-trimethylation promotes compaction of chromatin arrays through multimodal interactions. The results provide molecular insights into HP1α-mediated chromatin compaction and thus into the role of human HP1α in the regulation of gene silencing.

Multivalent Tau/PSD-95 interactions arrest in vitro condensates and clusters mimicking the postsynaptic density.

Alzheimer's disease begins with mild memory loss and slowly destroys memory and thinking. Cognitive impairment in Alzheimer's disease has been associated with the localization of the microtubule-associated protein Tau at the postsynapse. However, the correlation between Tau at the postsynapse and synaptic dysfunction remains unclear. Here, we show that Tau arrests liquid-like droplets formed by the four postsynaptic density proteins PSD-95, GKAP, Shank, Homer in solution, as well as NMDA (N-methyl-D-aspartate)-receptor-associated protein clusters on synthetic membranes. Tau-mediated condensate/cluster arrest critically depends on the binding of multiple interaction motifs of Tau to a canonical GMP-binding pocket in the guanylate kinase domain of PSD-95. We further reveal that competitive binding of a high-affinity phosphorylated peptide to PSD-95 rescues the diffusional dynamics of an NMDA truncated construct, which contains the last five amino acids of the NMDA receptor subunit NR2B fused to the C-terminus of the tetrameric GCN4 coiled-coil domain, in postsynaptic density-like condensates/clusters. Taken together, our findings propose a molecular mechanism where Tau modulates the dynamic properties of the postsynaptic density.

Determining the Physico-Chemical Composition of Biomolecular Condensates from Spatially-Resolved NMR.

Liquid-Liquid phase separation has emerged as fundamental process underlying the formation of biomolecular condensates. Insights into the composition and structure of biomolecular condensates is, however, complicated by their molecular complexity and dynamics. Here, we introduce an improved spatially-resolved NMR experiment that enables quantitative analysis of the physico-chemical composition of multi-component biomolecular condensates in equilibrium and label-free. Application of spatially-resolved NMR to condensates formed by the Alzheimer's disease-associated protein Tau demonstrates decreased water content, exclusion of the molecular crowding agent dextran, presence of a specific chemical environment of the small molecule DSS, and ≈150-fold increased concentration of Tau inside the condensate. The results suggest that spatially-resolved NMR can have a major impact in understanding the composition and physical chemistry of biomolecular condensates.

Molecular interactions of FG nucleoporin repeats at high resolution.

Proteins that contain repeat phenylalanine-glycine (FG) residues phase separate into oncogenic transcription factor condensates in malignant leukaemias, form the permeability barrier of the nuclear pore complex and mislocalize in neurodegenerative diseases. Insights into the molecular interactions of FG-repeat nucleoporins have, however, remained largely elusive. Using a combination of NMR spectroscopy and cryoelectron microscopy, we have identified uniformly spaced segments of transient ß-structure and a stable preformed α-helix recognized by messenger RNA export factors in the FG-repeat domain of human nucleoporin 98 (Nup98). In addition, we have determined at high resolution the molecular organization of reversible FG-FG interactions in amyloid fibrils formed by a highly aggregation-prone segment in Nup98. We have further demonstrated that amyloid-like aggregates of the FG-repeat domain of Nup98 have low stability and are reversible. Our results provide critical insights into the molecular interactions underlying the self-association and phase separation of FG-repeat nucleoporins in physiological and pathological cell activities.

Contributions of the N-terminal intrinsically disordered region of the severe acute respiratory syndrome coronavirus 2 nucleocapsid protein to RNA-induced phase separation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein is an essential structural component of mature virions, encapsulating the genomic RNA and modulating RNA transcription and replication. Several of its activities might be associated with the protein's ability to undergo liquid-liquid phase separation. NSARS-CoV-2 contains an intrinsically disordered region at its N-terminus (NTE) that can be phosphorylated and is affected by mutations found in human COVID-19 infections, including in the Omicron variant of concern. Here, we show that NTE deletion decreases the range of RNA concentrations that can induce phase separation of NSARS-CoV-2 . In addition, deletion of the prion-like NTE allows NSARS-CoV-2 droplets to retain their liquid-like nature during incubation. We further demonstrate that RNA-binding engages multiple parts of the NTE and changes NTE's structural properties. The results form the foundation to characterize the impact of N-terminal mutations and post-translational modifications on the molecular properties of the SARS-CoV-2 nucleocapsid protein. STATEMENT: The nucleocapsid protein of SARS-CoV-2 plays an important role in both genome packaging and viral replication upon host infection. Replication has been associated with RNA-induced liquid-liquid phase separation of the nucleocapsid protein. We present insights into the role of the N-terminal part of the nucleocapsid protein in the protein's RNA-mediated liquid-liquid phase separation.

Doxycycline Interferes With Tau Aggregation and Reduces Its Neuronal Toxicity.

Tauopathies are neurodegenerative disorders with increasing incidence and still without cure. The extensive time required for development and approval of novel therapeutics highlights the need for testing and repurposing known safe molecules. Since doxycycline impacts α-synuclein aggregation and toxicity, herein we tested its effect on tau. We found that doxycycline reduces amyloid aggregation of the 2N4R and K18 isoforms of tau protein in a dose-dependent manner. Furthermore, in a cell free system doxycycline also prevents tau seeding and in cell culture reduces toxicity of tau aggregates. Overall, our results expand the spectrum of action of doxycycline against aggregation-prone proteins, opening novel perspectives for its repurposing as a disease-modifying drug for tauopathies.

Effects of pharmacological modulators of α-synuclein and tau aggregation and internalization.

Parkinson's disease (PD) and Alzheimer's disease (AD) are common neurodegenerative disorders of the elderly and, therefore, affect a growing number of patients worldwide. Both diseases share, as a common hallmark, the accumulation of characteristic protein aggregates, known as Lewy bodies (LB) in PD, and neurofibrillary tangles in AD. LBs are primarily composed of misfolded α-synuclein (aSyn), and neurofibrillary tangles are primarily composed of tau protein. Importantly, upon pathological evaluation, most AD and PD/Lewy body dementia cases exhibit mixed pathology, with the co-occurrence of both LB and neurofibrillary tangles, among other protein inclusions. Recent studies suggest that both aSyn and tau pathology can spread and propagate through neuronal connections. Therefore, it is important to investigate the mechanisms underlying aggregation and propagation of these proteins for the development of novel therapeutic strategies. Here, we assessed the effects of different pharmacological interventions on the aggregation and internalization of tau and aSyn. We found that anle138b and fulvic acid decrease aSyn and tau aggregation, that epigallocatechin gallate decreases aSyn aggregation, and that dynasore reduces tau internalization. Establishing the effects of small molecules with different chemical properties on the aggregation and spreading of aSyn and tau will be important for the development of future therapeutic interventions.

Lysine/RNA-interactions drive and regulate biomolecular condensation.

Cells form and use biomolecular condensates to execute biochemical reactions. The molecular properties of non-membrane-bound condensates are directly connected to the amino acid content of disordered protein regions. Lysine plays an important role in cellular function, but little is known about its role in biomolecular condensation. Here we show that protein disorder is abundant in protein/RNA granules and lysine is enriched in disordered regions of proteins in P-bodies compared to the entire human disordered proteome. Lysine-rich polypeptides phase separate into lysine/RNA-coacervates that are more dynamic and differ at the molecular level from arginine/RNA-coacervates. Consistent with the ability of lysine to drive phase separation, lysine-rich variants of the Alzheimer's disease-linked protein tau undergo coacervation with RNA in vitro and bind to stress granules in cells. Acetylation of lysine reverses liquid-liquid phase separation and reduces colocalization of tau with stress granules. Our study establishes lysine as an important regulator of cellular condensation.